Platelet adhesion and aggregation at sites of vascular injury are key events required to arrest bleeding. However, the same haemostatic processes can also lead to thrombi within atherosclerotic arteries (i.e., coronary or cerebral arteries), which is the leading cause of morbidity and mortality worldwide. Unstable angina and myocardial infarction typically result from platelet adhesion and aggregation at the site of atherosclerotic lesions in coronary arteries. Antiplatelet therapies therefore provide a useful approach to mitigate platelet aggregation, thrombus formation, and hence, the risk of suffering a heart attack or stroke.
Currently available antiplatelet agents include ASPIRIN™, the thienopyridines (ticlopidine and clopidogrel), dipyridamole, and the platelet integrin αIIbβ3 (glycoprotein IIb/IIIa) receptor antagonists. The present invention relates to the latter category of antiplatelet agents, namely, the platelet αIIbβ3 receptor antagonists.
Integrin αIIbβ3, a calcium-dependent heterodimer containing a αIIb subunit and a β3 subunit, is the most abundant platelet adhesion receptor. There are 5-12×104 copies of αIIbβ3 expressed on each platelet, which accounts for approximately 17% of the total platelet membrane protein. It is a receptor for fibrinogen, fibronectin, vitronectin, von Willebrand factor, and thrombospondin, and mediates platelet aggregation, firm adhesion, and spreading. Mutations in either the αIIb or the β3 subunit have been found to result in Glanzmann thrombasthenia, an autosomal recessive bleeding disorder affecting platelet function (Bellucci, S., and Caen, J. (2002) Blood Rev. 16, 193-202).
The β3 subunit (GPIIa) of the complex is encoded by the ITGB3 gene, while the αIIb subunit (GPIIb) is encoded by the ITGA2B gene. Each subunit has been extensively studied individually, as the αIIbβ3 heterodimeric complex belongs to a class of cell adhesion molecule receptors that share common heterodimeric structures with alpha and beta subunits.
GPIIIa is the common β subunit of 2 integrins: αIIbβ3 complex and αvβ3 complex, which have distinctive alpha subunits (i.e., αIIb and αv). αvβ3 is also a receptor for fibronectin, vitronectin, von Willebrand factor, and thrombospondin. Both αIIbβ3 integrin and αvβ3 integrin are expressed on the platelet surface although the copies of αvβ3 integrin (2-4,000/platelet) are significantly fewer. Interestingly, αvβ3 integrin is also expressed on angiogenic endothelial cells and is required for new vessel development (angiogenesis) (Brooks P C et al, 1994. Science 264:569-571). Therefore, anti-β3 integrin antibodies may also have anti-angiogenic potential for tumor therapy.
Both integrin α and β subunits contribute to their ligand (e.g., fibrinogen, fibronectin) binding. The ligand binding pocket is formed by an N-terminal β propeller domain of the α subunit and the βA domain of the β subunit (top of FIG. 1). Three Ca2+ and one Mg2+ have been proposed in the β propeller domain of the α subunit; these divalent cations support one ligand binding site on this subunit. One divalent cation binding site has been proposed in the βA domain (vWF A domain-like domain) of the β subunit; this domain contains an RGD peptide binding site in the up-face of a Rossman fold structure. The RGD (arginine-glycine-aspartic acid) sequence in ligands (e.g., fibrinogen, vWF, fibronectin, vitronectin, prothrombin, etc.) is the recognition motif of the integrin family, and after ligand binding, some divalent cations may be replaced by an RGD portion of the ligands, but divalent cations are required to maintain the specific structure for ligand binding in most cases.
It is notable that many anti-PSI domain antibodies, if not all, may activate integrins by directly changing the integrin conformation. Based on earlier studies (Ni H et al, J Biol Chem. Apr. 3, 1998; 273(14):7981-7987), and the integrin structure model (Xiao T et al, Nature. Nov. 4, 2004; 432(7013):59-67), antibody binding to the PSI domain of integrin (e.g., β3 integrin) may enhance the “swinging out” of the leg of the β subunit and facilitate integrin-ligand binding. Therefore, anti-PSI domain of β3 integrin antibodies may lead to platelet activation and aggregation, and have therapeutic potential to stop bleeding.
Among the commercially available platelet αIIbβ3 receptor antagonists is the monoclonal antibody (mAb) 7E3, which binds to the αIIbβ3 receptor on the platelet. A humanized form of 7E3 is the active agent in the clinical drug REOPRO™ (abciximab), a drug developed for the treatment of thrombotic diseases. According to the REOPRO™ website, the drug blocks interaction of fibrinogen with αIIbβ3, and hence the final common pathway to platelet aggregation. REOPRO™ (abciximab) is thus claimed to block the formation of platelet aggregates and the formation of an arterial thrombus, and is used as an adjunct to percutaneous coronary intervention (PCI).
Another commercially available platelet αIIbβ3 receptor antagonist, tirofiban, is marketed as a drug in many countries under the brand name AGGRASTAT™. Tirofiban is a non-peptide αIIbβ3 receptor inhibitor, and is usually used in combination with heparin to help prevent blood clotting that occurs during certain heart conditions (e.g., acute coronary syndrome), or in medical procedures such as percutaneous coronary intervention (PCI).
Eptifibatide (INTEGRILIN™), another αIIbβ3 receptor antagonist, is an intravenous cyclical heptapeptide derived from a protein found in the venom of the southeastern pygmy rattlesnake (Sistrurus millarus barbouri). It has been shown to be efficacious in the treatment of patients during coronary angioplasty, myocardial infarction and angina.
A significant drawback of these αIIbβ3 antagonists, however, is the occurrence of side-effects, such as bleeding disorders. Due to the severity of these side effects, there is a need for continued research with a view towards developing new platelet aggregation inhibitors that can be safely used for antithrombotic therapy.